Polyamine is a substance present in most of the living cells. Spermidine or spermine belonging to polyamine is found in various species such as bacteria, fungus, and animals. Putrescine or 1,4-butanediamine which is a precursor in spermidine and spermine metabolism is found in a gram-negative bacteria or fungus, and it is present in wide range of concentration in various species suggesting that it has an important role in metabolic pathway.
Putrescine is a building block in a synthesis of polyamine nylon-4, 6 which is produced by reacting putrescine with adipic acid. To be used in manufacture of a processed plastic as a raw material, putrescine is usually produced by chemical synthesis involving conversion of propylene to acrylonitrile and to succinonitrile. This chemical synthesis consists of three-step process comprising catalytic oxidation reaction which consumes a lot of energy, reaction using a toxic chemical such as cyanide, and hydrogenation reaction that uses high-pressure hydrogen. Production of putrescine by chemical synthesis is not environmentally friendly and also consumes a lot of energy leading to depletion of petroleum resource. Therefore, a more environmentally friendly and energy-effective method involving biomass utilization needs to be developed for putrescine production.
In microorganism, a biosynthetic pathway of putrescine is the same as L-arginine biosynthetic pathway from glutamate to ornithine synthesis step. Putrescine can be synthesized by two pathways such as ornithine decarboxylation or arginine decarboxylation. These two pathways produce the energy required for metabolism or allow the cell to have resistance to oxidative stress. A method for production of putrescine at a high concentration by transformation of E. coli and Corynebacterium has been reported. Production of putrescine in E. coli can be achieved by increasing expression level of ornithine decarboxylase and glutamate acetyltransferase. Also, putrescine can be produced at high concentration by removing spermidine and acetylputrescine synthetic pathways which degrade or utilize putrescine (Qian. Z D. et al., Biotechnol. Bioeng. 104:4, 651-662, 2009, International Patent Publication No. WO06/005603. International Patent Publication No. WO09/125924). Meanwhile, in Corynebacterium sp. strain which lacks putrescine synthetic pathway, putrescine may be produced from ornithine through insertion of ornithine decarboxylase gene derived from E. coli or putrescine may be produced from L-arginine by insertion of the gene of L-arginine decarboxylase and agamatinase derived from E. coli. Ornithine pathway actually can produce about 50 times higher amount of putrescine than L-arginine pathway (Schneider et al., Appl. Microbiol. Biotechnol. 88:4, 859-868, 2010).
Meanwhile, it was found that E. coli can grow normal in the presence of 44 g/L putrescine, while Corynebacterium glutamicum can grow normal in the presence of 66 g/L putrescine. Therefore, it seems more effective to use Corynebacterium sp. strain which can survive at higher concentration of putrescine than E. coli in development of microorganism for producing putrescine.
Corynebacterium sp. strains are commercially applicable microorganism that is widely used in production of amino acid, nucleic acids, enzymes, and antibiotic analogs. In Corynebacterium sp. strain, L-arginine is synthesized from glutamate by enzymes expressed from the gene of arginine operon composed of argCJBDFRGH. Arginine operon genes that take the most important role in biosynthesis of L-arginine uses intracellularly synthesized L-glutamate as a substrate for arginine synthesis. FIG. 2 shows a schematic diagram of a synthetic pathway of arginine from glutamate in Corynebacterium sp. strain. In arginine synthetic pathway, ArgJ converts glutamate to N-acetylglutamate, ArgB converts N-acetylglutamate to N-acetylglutamyl phosphate, ArgC converts N-acetylglutamyl phosphate to N-acetylglutamate semialdehyde, ArgD converts N-acetylglutamate semialdehyde to N-acetylornithine, ArgJ converts N-acetylornithine to ornithine, ArgF converts ornithinie to L-citrulline, ArgG converts L-citrulline to argininosuccinate, and ArgH converts argininosuccinate to arginine.
Previously known arginine-producing strains were developed by increasing the expression level of enzyme involved in arginine biosynthesis through introducing mutation to arginine operon or mutating promoter. Among the genes in arginine operon, argR which regulates and suppresses the expression of arginine operon gene and argB which is inhibited by arginine concentration have been targeted in many studies to increase arginine production level (Korea Patent Publication No. 2010-0060909).
Putrescine biosynthetic pathway is the same as arginine biosynthetic pathway from glutamate to ornithine synthesis step. Then putrescine is produced from the synthesized ornithine by ornithine decarboxylase (ODC). Therefore, in order to prepare a strain capable of producing high amount of putrescine, a sufficient amount of ornithine has to be made first. When glutamate was added to the argF- and argR-deleted strain of wild-type E. coli W3110, ornithine production level was increased by 20%. Also in addition to the pathway from glutamate to ornithine, when the pathway from glutamate to proline synthesis was blocked by knocking out proB gene which encodes γ-glutamylkinase involved in the first step thereof, the ornithine production level was increased as well. This suggests that when the intracellular level of glutamate is increased, it has positive effects on ornithine production in the cell. In a study of high yield production of glutamate which is a precursor of ornithine, Corynebacterium glutamicum has been studied for a long time. In this regard, it has been reported that the glutamate exporting activity of Corynebacterium glutamicum is enhanced when the cell lacks biotin or when the cell is treated with penicillin G or fatty acid ester surfactant. This result suggests that when the cell wall is damaged, glutamate can be exported better through cytoplasm.
NCgl1221 protein derived from Corynebacterium glutamicum wild-type strain (ATCC 13032) is known to promote the betain export and has a similar amino acid sequence as that of yggB which codes for a mechanosensitive channel protein (Korea Patent Publication No. 2010-0017581).